Manipulating genomes

• Polymerase Chain Reaction 


  In-vitro method of amplifying small fragments of dna that you are interested in so you have enough to analyse
• Example of use of PCR...

  Dna from drop of blood at crime scene so you can identify murderer/ victim
• Where is PCR usually carried out?

  In thermocycler which periodically changes temp so you don't have to do it manually
• What do you add to the thermocycler in the first step

  1. dsDNA fragment
  2. Free DNA nucleotides
  3. Taq DNA polymerase
  4. Primers
• What are primers 


  Small ssDNA fragments (around 20-24 bases long) that are complementary to the first few bases of the DNA fragments or part of the fragment you want to amplify
• Why are primers with 60%+ C or G used?

  Because they can form 3H bonds and anneal to DNA fragments more strongly
• What is the 2nd step in PCR

  1. Mixture is heated to 95c to break H bonds holding 2 strands of dsDNA fragment together (denaturation)
  2. Taq DNA doesn't denatured at this temp as it's obtained from thermophilic bacteria so it's thermostable
• What is the 3rd step of PCR

  1. Mixture is cooled to 65c which allows primers to anneal to beginning of 2 ssDNA fragments
  2. Anneals to 3' end of each strand
  3. Promotes a dsDNA section that Taq DNA polymerase can bind to
• What is the 4th step of PCR

  1. Temp is increased to 72c as its the optimum temp for Taq DNA polymerase
  2. Taq DNA polymerase catalyses formation of phosphodiester bonds between new nucleotides in 5' -> 3' direction on new strand
• What is the 5th step of PCR

  1. When Taq DNA polymerase reaches end of DNA fragment, it will detach and first cycle is complete
  2. Results in 2 copies of dsDNA fragment
  3. Cycle is repeated continuously so no. of dsDNA fragments increases exponentially